The present invention relates to in-situ gelation of a pectic substance. Specifically, the invention relates to a pectin in-situ gelling formulation for the delivery and sustained release of a physiologically active agent to the body of an animal. More specifically, the pectic substance is derived from Aloe vera L. plant.
Abbreviations Used Herein Include:
CMC, carboxylmethyl cellulose; Da, dalton; DM, degree of methylation; Gal A, galacturonic acid; HEC, hydroxyethyl cellulose; HM, high methoxyl; HPMC, hydroxypropylmethylcellulose; kDa, kilodaltons; LM, low methoxyl; PBS, phosphate buffered saline; PEG-PLGA-PEG, polyethylene glycol-poly(lactic-co-glycolic acid)-polyethylene glycol; PEO-PLLA, poly(ethylene oxide)-poly(L-lactide); PEO-PPO-PEO, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide).
Pectin is a biodegradable acidic carbohydrate polymer. Pectin is commonly found in plant cell walls. The cell wall of a plant is divided into three layers consisting of the middle lamella, the primary wall and the secondary cell wall. The middle lamella is richest in pectin. The chemistry and biology of pectin have been extensively reviewed (Pilnik and Voragen, Advances in plant biochemistry and biotechnology 1, 219-270, 1992; Voragen et al, In Food polysaccharides and their applications. pp 287-339. Marcel Dekker, Inc. New York, 1995; Schols and Voragen, In Progress in Biotechnology 14. Pectins and pectinases, J. Visser and A. G. J. Voragen (eds.). pp. 3-20. Elsevier Science Publishers B. V. Amsterdam, 1996).
Pectin consists of an xcex1-(1xe2x86x924)-linked polygalacturonic acid backbone intervened by rhamnose residues and modified with neutral sugar side chains and non-sugar components such as methyl and acetyl groups. The extent of rhamnose insertions and other modifications vary depending on plant sources. The Gal A content is generally more than 70% whereas the rhamnose content is typically  less than 2%. Rhamnose residues are xcex1-(1xe2x86x922)-linked to Gal A residues in the backbone. They cause the formation of a T-shaped kink in the backbone chain, and the increase in rhamnose content leads to more flexible molecules. The neutral sugar side chains are attached to the rhamnose residues in the backbone at the O-3 or O-4 position. The rhamnose residues tend to cluster together on the backbone. Hence, this region with side chains attached is referred to as the xe2x80x9chairy regionxe2x80x9d while the rest of the backbone is named the xe2x80x9csmooth region.xe2x80x9d
Methylation occurs at carboxyl groups of Gal A residues. The degree of methylation or methyl-esterification (xe2x80x9cDMxe2x80x9d) is defined as the percentage of carboxyl groups (Gal A residues) esterified with methanol. Based on the DM, pectins are divided into two classes, low methoxyl (xe2x80x9cLMxe2x80x9d) pectin with a DM of  less than 50% and a high methoxyl (xe2x80x9cHMxe2x80x9d) pectin with a DM of  greater than 50%. Commercial pectins derived from citrus and apples are naturally HM pectins. LM pectins are typically obtained through a chemical de-esterification process. Commercial LM pectins typically have a DM of 20-50%. A completely de-esterified pectin is referred as xe2x80x9cpectic acidxe2x80x9d or xe2x80x9cpolygalacturonic acidxe2x80x9d. Pectic acid in the acid form is insoluble but is soluble in the salt form. The common salt form of pectic acid is either sodium or potassium.
Pectin is most stable at acidic pH levels between approximately 3-4. Below pH 3, methoxyl and acetyl groups and neutral sugar side chains are removed. Under neutral and alkaline conditions, methyl ester groups are saponified and the polygalacturonan backbone breaks through xcex2-elimination-cleavage of glycosidic bonds on the non-reducing ends of methylated Gal A residues. Pectic acids and LM pectins are relatively more resistant to neutral and alkaline conditions since there are only limited numbers of methyl ester groups or none at all.
Current commercial pectins are mainly from citrus and apples. However, besides citrus and apples, pectins can also be isolated from many other plants. All vegetables and fruits that have been examined contain pectins. Pectins from sugar beets, sunflowers, potatoes, and grapefruits are just a few other well known examples.
Both HM and LM pectins form gels. However, these gels form via totally different mechanisms (Voragen et al, In Food polysaccharides and their applications. pp 287-339. Marcel Dekker, Inc. New York, 1995). HM pectin forms a gel in the presence of high concentrations of co-solutes (sucrose) at low pH. LM pectin forms a gel in the presence of calcium, thus, it is xe2x80x9ccalcium-reactive.xe2x80x9d The calcium-LM pectin gel network is built by formation of what is commonly referred to as an xe2x80x9cegg-boxxe2x80x9d junction zone in which Ca++ causes the cross-linking of two stretches of polygalacturonic acid chains.
HM pectins are generally not reactive with calcium ions and therefore cannot form a calcium gel. However, certain HM pectins have been reported to be calcium sensitive and capable of calcium gel formation. In addition, HM pectins can be made calcium-reactive by a block wise de-esterification process while still having a DM of  greater than 50%. See, Christensen et al. U.S. Pat. No. 6,083,540.
Calcium-LM pectin gel formation is influenced by several factors, including DM, ionic strength, pH, and molecular weight (Garnier et al., Carbohydrate Research 240, 219-232, 1993; 256, 71-81, 1994). The lower the DM and the higher the molecular weight, the more efficient the gelation. Furthermore, the calcium-LM pectin gelation is more efficient at a neutral pH of xcx9c7.0 than xcx9c3.5. Lastly, the addition of monovalent counter ion (NaCl) enhances the gelation, i.e., less calcium is required for gel formation.
Pectins are typically utilized in the food industry and classified by the FDA as xe2x80x9cGRASxe2x80x9d (Generally Regarded As Safe). They have also long been used as colloidal and anti-diarrhea agents. Recently, pectins have been utilized in the areas of medical device and drug delivery (Thakur et al., Critical Reviews in Food Science and Nutrition 37, 47-73, 1997). In the case of drug delivery, pectin has found its presence in many experimental formulations for oral drug delivery to the colon because pectin is readily degraded by bacteria present in this region of the intestines. The pectin is either used directly with no gelation involved or a pectin calcium gel is preformed to encapsulate the drug agent before administration. Ashford et al., J. Controlled Release 26, 213-220, 1993; 30, 225-232, 1994; Munjeri et al., J. Controlled Release 46, 273-278, 1997; Wakerly et al., J. Pharmacy and Pharmacology 49, 622-625, 1997; International Journal of Pharmaceutics 153,219-224,1997; Miyazaki et al., International Journal of Pharmaceutics 204, 127-132, 2000. Prior to the present invention, there appears to be no attempt made to examine the in-situ gelling ability of pectins.
Aloe pectin isolated from Aloe vera plant as described in U.S. Pat. No. 5,929,051, the entire content of which is incorporated herein by reference. It is naturally a LM pectin and capable of calcium gelation. In addition, it possesses several unique chemical properties that are particularly related to gelation, including a high molecular weight ( greater than 1xc3x97106 Da), a high Gal A content (as high as  greater than 90%), and a low DM ( less than 10%).
Current commercial pectins typically have a size of 7-14xc3x97104 Da and Gal A content of xcx9c75% (Voragen et al, In Food polysaccharides and their applications. pp 287-339. Marcel Dekker, Inc. New York, 1995). These pectins have a rhamnose content of  less than 2%. Commercial LM pectins and other natural LM pectins have a DM of  greater than 20%. A DM below 10% makes Aloe pectin nearly a pectic acid. A pectin with such a low DM, a high molecular weight, and a high Gal A content has not been described previously. Aloe pectin is an off white powder as the finished product, whereas all current commercial and experimental pectins are yellow to tan powders.
Drug delivery has been a subject of intense studies over recent years. The goal is to achieve sustained (or slow) and/or controlled drug release and thereby improve efficacy, safety, and/or patient comfort. A sustained and/or controlled release of the drug agents is achieved by the retardation of drug diffusion by and/or gradual disintegration of the polymer matrix following application.
In-situ gelation is a process of gel formation at the site of application after the composition or formulation has been applied to the site. In the field of human and animal medicine, the sites of application refers to various injection sites, topical application sites, surgical sites, and others where the agents are brought into contact with tissues or body fluids. As a drug delivery agent, the in-situ gel has an advantage related to the gel or polymer network being formed in-situ providing sustained release of the drug agent. At the same time, it permits the drug to be delivered in a liquid form.
Polymers capable of in-situ gelation have been described. They include Poloxamer, Pluronics (Vadnere et al., Int. J. Pharm., 22, 207-218, 1984), various copolymers such as PEO-PLLA and PEG-PLGA-PEG (Jeong et al., Nature 388, 860-862, 1997; Jeong et al., J. Controlled Release 63, 155-163, 2000), cellulose acetophalate latex (Gurny et al. J. Controlled Release 353-361, 1985), Gelrite (Rozier et al., Int. J. Pham. 57, 163-168, 1989), Carbopol, and Matrigel. The gel formation is induced by temperature change (Poloxamer, Pluronics, PEO-PLLA diblock copolymer, PEG-PLGA-PEG triblock copolymer, and Matrigel), pH change (cellulose acetophalate latex and Carbopol), or reaction with mono- or di-valent cations (Gelrite). However, most of them require a high polymer concentration for in-situ gel formation ( greater than 20%) (Poloxamer, PEO-PLLA diblock copoly, PEG-PLGA-PEG triblock copolymer, cellulose and acetophalate latex). The thermally gelling polymers (Poloxamer, Pluronics, PEO-PLLA diblock copolymer, PEG-PLGA-PEG triblock copolymer, and Matrigel) also have the disadvantage of gelling before administration due to temperature change during packaging or storage. Unfortunately some of these polymers are not biodegradable such as Poloxamer or require manipulation of the temperature before administration (PEO-PLLA diblock copolymer) or during formulation (Pluronics and Gelrite). An ophthalmic in-situ gelling drug delivery formulation consisting of a mixture of Carbopol and Pluronic was found to be more effective than formulations consisting of either one. However, Pluronic is used at 14% (Lin and Sung, Journal of Controlled Release 69, 379-388, 2000). Such polymers are therefore not well suited for medical applications in humans and animals. Furthermore, many of these polymers form only a hydrogel which is a viscous but still flowing solution (e.g., Poloxamer and Pluronics).
The in-situ gelation compositions using ionic polysaccharides have been disclosed in U.S. Pat. No. 5,958,443, which consist of a drug, a polymer and a gel forming ionic polysaccharide which consist of two components, an ionic polysaccharide and a cross-linking ion capable of cross-linking the former. The in-situ gel formation is induced by the application of the cross-linking ions.
Thus, a great need exists for a simpler and more efficient in-situ gelling composition that employs only a low polymer concentration for the purposes of drug delivery.
One embodiment of the present invention pertains to using a pectic substance to provide a biodegradable in-situ gelling composition for animal and human use. The composition transforms from a liquid into a gel following administration to the target site. Preferably the pectic substance is Aloe pectin.
One object of the present invention is to provide a composition for controlled, or sustained, release of a physiologically active agent in the body of an animal.
Another object of the present invention is to provide for a transparent polymer solution wherein no dramatic increase in gel cloudiness is created beyond certain concentration ranges. Preferably the composition is capable of creating an in-situ gel at low concentrations.
Another object of the present invention is to provide for a transparent polymer solution wherein a thickener is added. Preferably the composition is capable of creating an in-situ gel at low concentrations.
A further object of the present invention is to provide for a composition that is capable of creating an in-situ gel at low concentrations once delivered in the liquid form.
Another object of the present invention is to provide for a composition for drug delivery. In the case of drug delivery, for example, a therapeutic or diagnostic agent is incorporated into the formulation or composition. These agents can be small molecules as well as large ones such as proteins. Preferably the composition is capable of forming an in-situ gel at low concentrations.
These and other objects of the present invention are provided by the described embodiments of the present invention. The foregoing discussion has outlined some of the more pertinent features of the present invention. These should be construed to be merely illustrative of some of the more prominent features and applications of the invention. Accordingly, a fuller understanding of the invention maybe had by referring to the following Detailed Description.